MEK inhibition reduced vascular tumor growth and coagulopathy in a mouse model with hyperactive GNAQ

Activating non-inherited mutations in the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) gene family have been identified in childhood vascular tumors. Patients experience extensive disfigurement, chronic pain and severe complications including a potentially lethal coagulopathy termed Kasabach-Merritt phenomenon. Animal models for this class of vascular tumors do not exist. This has severely hindered the discovery of the molecular consequences of GNAQ mutations in the vasculature and, in turn, the preclinical development of effective targeted therapies. Here we report a mouse model expressing hyperactive mutant GNAQ in endothelial cells. Mutant mice develop vascular and coagulopathy phenotypes similar to those seen in patients. Mechanistically, by transcriptomic analysis we demonstrate increased mitogen activated protein kinase signaling in the mutant endothelial cells. Targeting of this pathway with Trametinib suppresses the tumor growth by reducing vascular cell proliferation and permeability. Trametinib also prevents the development of coagulopathy and improves mouse survival.


Vascular membrane marker would be useful in
relate ERK activity to vessel identity (artery/capillary/vein) and anatomy (tuft/normal). 5. The authors see vascular tuft formation in adult GNAQ Q209L mice 4 days after tamoxifen injection (Fig 4). It is expected to take at least 1 day to allow for sufficient amount of mutant protein to be expressed, thus it is unexpected that a severe phenotype develops in the adult vasculature in such a short time frame. I am wondering if the vascular phenotype observed in these mice reflects tamoxifenindependent leakage leading to transgene expression and vascular expansion already at an earlier developmental stage. Cre leakage has been reported to occur in both lines used in the study (PMID: 31641921). What is the genotype of the control mice, are they GNAQ Q209L Cre+ mice that were not treated with tamoxifen, or Cre-mice?
6. Related to the previous comment, it is also very surprising that the authors see hemostatic complications including low platelet numbers and elevated D-dimer levels in adult GNAQ Q209L just 5 days after tamoxifen injection (Fig 7). Have the authors excluded a potential targeting of and direct effect on hematopoietic cells? I believe that Kasabach-Merritt phenomenon is thought to result from consumption of platelets and fibrinogen by intralesional thrombosis. Please provide supporting evidence for intralesional thrombosis, and exclude the effect of tamoxifen-independent leakage by providing the analysis of untreated control mice carrying both GNAQ Q209L and Cre alleles. 7. Considering the potential tamoxifen-independent recombination discussed above, MEK inhibitor experiments should also include untreated control mice carrying the GNAQ Q209L and Cre alleles. Bright-field images (such as shown in Fig 4E) and/or lower magnification immunofluorescence images should be shown. In addition, it would be important to assess the effect of MEK inhibition on already established lesions (i.e. can this lead to the regression of lesions).
8. Increased pERK and KI67 staining in a human vascular tumor tissue in Fig 6 provides additional support for the involvement of ERK in GNAQ-driven pathology, and confirms previous observations (PMID: 30112971), but only 1 sample is analyzed. The comparison of vascular parameters in vascular tumor and human foreskin is not appropriate, considering that these tissues have different organization of the vasculature and represent different anatomic location. Since vascular tumors are known to be proliferative and may show increased pERK and KI67 independent of GNAQ activity, additional analysis of GNAQ-driven capillary malformations would be helpful.
Minor comments: 1. The scheme shown in Fig 1A and B is confusing. Please indicate for each experiment which Cre line and tamoxifen administration protocol was used.
2. The genotype of the control mice should be specified.
3. Some of the information is repetitive; for example, Fig 2 and 3 could be combined.
4. Line 162, 163: 'Two-photon microscopy and Imaris image analysis showed the presence of perfused vascular tufts (see arrows) in the brain (Fig.1E).' Non-perfused tufts would not be visualized, but immunofluorescence analysis of brain sections after an injection with a fixable dextran could demonstrate that all lesions are perfused.
Reviewer #2 (Remarks to the Author): The authors established two mouse models in activating Gaq11 or overexpressing GNAQR183A specifically in vascular endothelial cells. From the phenotype analyses, ERK1/2 activation and MEK inhibitor therapeutics, it was clearly demonstrated that GNAQ plays an essential role in vascular development and homeostasis.
General and major concerns: The endothelial cell-specific activation and expression were not characterized. In general, most of figures on microvessels lack high power images. Phenotypes of mice were not analyzed thoroughly, for example, the gross growth, and survival rate, and physiological changes (e.g why early lethal if it was, which was mentioned in the discussion). The work also lacks novel mechanistic insights into how GNAQ cause vascular malformations and solely selected ERK1/2 for study. Therefore, the study was considered as mouse model with phenotypic verifications.
The phenotype characterization: it was limited to a very short time frame (3-5 days after induction) without clear rationale. Any late time points were analyzed? Some specific concerns (could be applied to all figures): Fig.1: Two photon imaging was nice. A dynamic video would help to visualize perfusion and flow within the tufts. Fig. 2: B/C: Quantifications of vessel density in retinas was missing. Section imaging of retinas and hyaloid would have better visualize the tufts. Vessel diameters: were they artery, vein or microvessels? Were microphage number differed in KO groups vs WT? Some tissues were examined (from Fig.1-5). However, the study was simply vascular densities. No functional studies were performed. Fig.6 had human speciesmen were strength. Fig.7 therapeutics with MEK inhibitor was nice. It would be more informative to dissect why ERK1 and if GNAQ inhibitors had better efficacy in the mouse models.
Reviewer #3 (Remarks to the Author): In this manuscript, Schrenk et al. generated the first murine model for GNAQ-related vascular anomalies, exhibiting endothelial cell (EC)-specific expression of the constitutively active mutant GNAQ pQ.209L of the Gαq protein. Gαq gain-of-function mutation results in widespread abnormal vascular morphogenesis and potential thrombocytopenia and severe coagulopathy. Such vascular morphogenesis defects are consistent with findings in patients with Gαq gain-of-function mutations. The authors characterized vascular abnormalities in the brain, retina, hyaloid, subcutaneous muscle and intestinal muscle in GNAQQ209 mice at different time points of development, and confirmed MAPK/ERK activation and proliferation markers in patient-derived tissue. Finally, the authors performed proof-of-concept experiments, demonstrating that MEK inhibition with Trametinib rescued vascular and coagulopathy phenotypes.
Since mechanisms by which Gαq regulates aberrant vascular development and remodeling remain unknown, this paper provides a murine model for studying (EC-specific) GNAQ-related vascular anomalies that will aid in development of targeted therapies, which are currently lacking. The authors provide extensive evidence of aberrant vascular morphologies that recapitulate what are seen in patients. The manuscript is easy to follow, and figures containing schematics of mouse breeding and experimental timeline are helpful. However, there are important limitations in this manuscript that limit enthusiasm that are listed below as well as more minor concerns.
MAJOR CONCERNS 1. This study is very observational. There was no further insights into the functionality of the altered vasculature. How does mutant Gαq affect EC biology beyond EC proliferation? Is the endothelium more inflamed? 2. There are very important controls not done in this manuscript that are needed to complete the claim that an EC-specific overexpression of GαqQ209 is responsible for the observed vascular effects. These include showing that there was no leakage of expression until tamoxifen was given. The controls in Figs 2-8 were done in mice that were littermates but did not have the GαqQ209 expression vector. It should have been done in mice with the GαqQ209 construct but without tamoxifen. Were the vessels normal until that was done? Also were the non-ECs normal and the effect was targeted to the endothelium? Finally, what was the relative level of GαqQ209 pre and post tamoxifen versus native Gαq? Was the effect seen just because there is a massive increase in Gαq in the ECs and even normal Gαq would have resulted in the same outcome. 3. There were two different EC-specific promoters studied. The rationale for that and why some studies were done mixing the two or with only one is not explained other than one statement that the results were not different, yet we don't know efficiency of the two in driving human GαqQ209 expression in different tissues.
MINOR CONCERNS 1. The authors should discuss Huang et al. ATVB 2022. (PMID: 34670408) on EC biology and its support of the studies in this manuscript. 2. A report of complete blood count and coagulation tests (PT and aPTT) in these mice as well as blood smears to look for schistocytes/polychromasia would be helpful. The effects of Trametinib on the blood counts should be compared to the non-tamoxifen-treated control discussed above as well as wildtype mice. 3. Page 6 -line 193: please explain the difference in subcutaneous tissue vascular area in early onset versus adult onset of GNAQ mutation. Why is it that only in early onset of GNAQ mutation that the authors observed no change in subcu. vascular area? 4. How does mutant Gαq affect EC biology beyond cell proliferation? Is the endothelium more inflamed? Do EC express higher levels of surface adhesion molecules? What about vascular permeability? Are there any changes in blood pressure longeviety? 5. Please provide evidence of thrombosis in the vasculature. For example, fibrin & platelet deposition at the lesions? 6. Trametinib also inhibits platelet MEK. Is the observed reverse of thrombocytopenia and coagulopathy seen in Trametinib-treated iEC-GNAQQ209L mice due to direct effect of Trametinib on platelets and other cells? Please discuss. 7. Consider changing the title of the manuscript? The current title states what is already known, not really what the authors are trying to show. 8. Since both male and female mice were used, did the author observe any sex difference? 9. An edited manuscript will be returned, but minor typos include: Use hyphen when appropriate. "Ki67-postive" not "Ki67 positive". Double check µm versus µM. Some units are mixed up. Subcutaneous "tissue", not subcutaneous "muscle". Use µL, not uL. Check spacing consistency throughout. For example, area≥ 200µm2 vs. area ≥ 200µm2. Mean ± SD vs. mean±SD.

RESPONSE TO REVIEWERS
Reviewer #1 (Remarks to the Author): Schrenk et al present a novel mouse model for GNAQ-related vascular anomalies. Inducible EC-specific expression of a gain-of-function GNAQ Q209L at different developmental stages or in adult mice led to vascular lesions characterized by enlarged vessels, and increased EC proliferation. In line with previous studies in cultured ECs and melanoma cells, GNAQ Q209L expression in mouse endothelium also led to increased ERK activation. Finally, the authors show that the formation of vascular lesions could be inhibited by a treatment with a MEK inhibitor.
The novelty of this study relates to the generation and characterization of the first genetic mouse model for GNAQrelated vascular pathology. The study remains, however, descriptive and does not provide new mechanistic insight into the associated disease mechanisms. It is of interest that GNAQ mutations are causative of hemangiomas and capillary malformations. It is not clear if the model generated in this study recapitulates the pathophysiology of these two distinct disease entities but it could potentially be used to address clinically important questions on their etiology and pathogenesis. R: We thank the reviewer for the feedback on our study. We have extensively revised our manuscript to address the major points raised and provide novel mechanistic insights on GNAQ Q209L-related vascular tumors.
Major comments: 1. The authors' conclusion that 'Gaq hyperactivation in EC induces vascular defects that are reminiscent of vascular anomalies such as CM/SWS' is not fully supported by data ( Fig S1). They show increased vascular density based on staining of sections of facial skin, but why was the phenotype not analyzed in the retina, as done in Fig 2? The authors instead study hyaloid vessels that regress postnatally. The phenotype shown in Fig S1D could reflect lack of hyaloid vessel regression rather than increased vascularity. This data does not seem relevant to the current study.
R: We agree with this comment. In this revised manuscript we have removed the hyaloid data and the link with CM/SWS. Data in the new Suppl. Fig.S3 show increased vascularity in the skin of mutant mice expressing the endothelial-specific hyperactive Gaq with the use of hM3Dq system -this data is supportive of our findings with the GNAQ Q209L model.
2. The analysis of the vascular phenotype is superficial, and mainly limited to the assessment of vascular tuft area. Quantification of tuft area over total area (Fig 1D, Fig 2C) will always show significant increase in the mutant since the control group does not have any tufts. This quantification may therefore not be meaningful.

R:
The vascular tufts are the main histopathological hallmark of GNAQ-related vascular anomalies, and it is important to document their number and size as this will enable preclinical studies with the use of pharmacological therapies. In this substantially revised manuscript, in addition to the tuft analysis, we have extended our characterization of the vascular defects which now includes several parameters: 1-vascular density, 2-diameter, 3-vessel size distribution, 4-number and 5-area of tufts. Furthermore, to increase the relevance of our study, we have characterized vascular permeability (Fig.3, Suppl. Video 3), coagulopathy (Fig.4, Suppl. Fig.S11-13,  Suppl. Video 4), and transcriptomic analysis of the GNAQ-mutant EC (Fig.5, Suppl. Fig.S14).
3. Are only certain vascular beds/organs or vessel types (artery/capillary/vein) sensitive to the activation of GNAQ, and does this mimic human disease pathology? Consistent analysis of different organs at different developmental stages should be provided.
R: This is a good question. GNAQ-related vascular anomalies primarily affect capillaries and veins. In this revised manuscript we utilized the developing vasculature of the retina as a model in which arteries and veins can be readily distinguished. Analysis of mutant iEC-GNAQ Q209L mice at P8 showed that vascular tufts were exclusively localized in retinal veins and capillaries, while absent in the arteries (see new Suppl. Fig.S5). Fig 3 to relate ERK activity to vessel identity (artery/capillary/vein) and anatomy (tuft/normal).

R:
We agree this would a great experiment, however pERK staining did not work in whole mounts neither in flat mount retinas. To address this point, we now show EdU staining labeling proliferative cells in whole mount intestinal muscularis and show that EdU+ ECs are located in the vascular tufts (Fig.2H-J and Suppl Video 2). Furthermore, we have analyzed EC proliferation in the vasculature of Trametinib-treated mice and see a significant reduction of EdU+ EC compared to vehicle-treated mice (Fig. 7G, Suppl Video 5, and Suppl. Fig. S15), confirming the importance of ERK activity in EC hyperproliferation and tuft formation.

5.
The authors see vascular tuft formation in adult GNAQ Q209L mice 4 days after tamoxifen injection (Fig 4). It is expected to take at least 1 day to allow for sufficient amount of mutant protein to be expressed, thus it is unexpected that a severe phenotype develops in the adult vasculature in such a short time frame. I am wondering if the vascular phenotype observed in these mice reflects tamoxifen-independent leakage leading to transgene expression and vascular expansion already at an earlier developmental stage. Cre leakage has been reported to occur in both lines used in the study (PMID: 31641921). What is the genotype of the control mice, are they GNAQ Q209L Cre+ mice that were not treated with tamoxifen, or Cre-mice? R: We thank the reviewer for bringing up this point so we can increase the rigor of our study. In this revised manuscript we now include no-tamoxifen mutant mice controls for most of the experiments and highlight in each graph the specific genotype of mice used for each experiment. Pups and young adults mutant mice that did not receive tamoxifen had a phenotype comparable to WT mice. Additionally, we have included comprehensive analysis of efficiency and specificity of the Cdh5-iCreER T2 driver with and without tamoxifen, by crossing GNAQ Q209L mice with the Rosa26 tdTomato lineage reporter. There was only minimal recombination in Chd5-iCreERT2; tdTomato mice that were not administered with tamoxifen (1.8±088% in pups; 2.7±2.50% and 2.20±1.60% in adult mice subcutaneous and intestinal muscularis tissues) (see Suppl. Fig.S2 and S8). We have also analyzed survival of mutant mice that did not receive tamoxifen, and at 13 months mice are alive and do not have overt phenotype or illness.
6. Related to the previous comment, it is also very surprising that the authors see hemostatic complications including low platelet numbers and elevated D-dimer levels in adult GNAQ Q209L just 5 days after tamoxifen injection (Fig 7). Have the authors excluded a potential targeting of and direct effect on hematopoietic cells? I believe that Kasabach-Merritt phenomenon is thought to result from consumption of platelets and fibrinogen by intralesional thrombosis. Please provide supporting evidence for intralesional thrombosis, and exclude the effect of tamoxifen-independent leakage by providing the analysis of untreated control mice carrying both GNAQ Q209L and Cre alleles. Suppl. Fig. S13). 2-We now provide evidence of platelet trapping and intralesional fibrinogen accumulation in the vascular tufts (Fig.4, Suppl. Fig.S12 and Video 4). Fig. 4 and Suppl. Figs.S10).

3-Furthermore, we now include data on complete cell counts (CBC) including platelets and RBC, and D-dimer for no-tamoxifen mutant controls and tamoxifen-treated Cre-allele controls (see
7. Considering the potential tamoxifen-independent recombination discussed above, MEK inhibitor experiments should also include untreated control mice carrying the GNAQ Q209L and Cre alleles. Bright-field images (such as shown in Fig 4E) and/or lower magnification immunofluorescence images should be shown. In addition, it would be important to assess the effect of MEK inhibition on already established lesions (i.e. can this lead to the regression of lesions). R: 1. To address the reviewer's question we included data on untreated (no tamoxifen) control mice carrying the GNAQ Q209L and the Cdh5-CreER T2 alleles in Figs.1, 2 and 4. These do not show a vascular phenotype. The potential effects of Trametinib on control mice was investigated in the therapeutic scheme (see Fig.7N) and these mice lived until the endpoint of the experiment. 2. In this revised manuscript we have included brightfield images of the subcutaneous and intestinal tissue for the Trametinib treatment experiments. 3. We have performed a therapeutic treatment scheme with Trametinib on already formed lesions. For this experiment we have used a lower dose of tamoxifen to extend the life span of the mice. Daily Trametinib treatment started 8 days after tamoxifen induction, and significantly extended the life span of the mutant mice (see Fig.7N). Fig 6 provides additional support for the involvement of ERK in GNAQ-driven pathology, and confirms previous observations (PMID: 30112971), but only 1 sample is analyzed. The comparison of vascular parameters in vascular tumor and human foreskin is not appropriate, considering that these tissues have different organization of the vasculature and represent different anatomic location. Since vascular tumors are known to be proliferative and may show increased pERK and KI67 independent of GNAQ activity, additional analysis of GNAQ-driven capillary malformations would be helpful.

R: We have only 1 patient sample with confirmed GNAQ p.Q209L mutation and it is not possible to access control tissue from the desired anatomic location in human subjects. We think the use of neonatal foreskin from different donors should be acceptable as control as it is a proliferative and highly vascularized tissue. Furthermore, data has been carefully normalized by EC number to account for the increased EC number in the vascular tumor. We understand the Reviewer's point about the specificity of increased ERK signaling in EC expressing the GNAQ p.Q209L mutation. For this reason, we now include data on RNA sequencing of EC expressing GNAQ-Q209L and GNAQ-WT. MAPK signaling pathway is one of the top differentially regulated KEGG and GO-BP pathway. We generated EC lines by transducing them with lentiviral constructs promoting Doxycycline-inducible (i) expression of GNAQ-Q209L and GNAQ-WT. ERK signaling was increased only in the mutant EC GNAQ-Q209L, in a Doxycycline dose-dependent manner (see new Fig.5F).
Minor comments: 1. The scheme shown in Fig 1A and B is confusing. Please indicate for each experiment which Cre line and tamoxifen administration protocol was used. R: We included breeding scheme and Cre-driver used in each Figure. 2. The genotype of the control mice should be specified. R: We specified the genotype for all of the control and mutant mice with color-coded labels.
3. Some of the information is repetitive; for example, Fig 2 and 3 could be combined. R: This manuscript has been extensively revised and these Figs are now supplemental.
4. Line 162, 163: 'Two-photon microscopy and Imaris image analysis showed the presence of perfused vascular tufts (see arrows) in the brain (Fig.1E).' Non-perfused tufts would not be visualized, but immunofluorescence analysis of brain sections after an injection with a fixable dextran could demonstrate that all lesions are perfused. R: Thank you for this suggestion. We have corrected this in the Result section for new Suppl. Fig.S4.
Reviewer #2 (Remarks to the Author): The authors established two mouse models in activating Gaq11 or overexpressing GNAQR183A specifically in vascular endothelial cells. From the phenotype analyses, ERK1/2 activation and MEK inhibitor therapeutics, it was clearly demonstrated that GNAQ plays an essential role in vascular development and homeostasis.
General and major concerns: The endothelial cell-specific activation and expression were not characterized. In general, most of figures on microvessels lack high power images. Phenotypes of mice were not analyzed thoroughly, for example, the gross growth, and survival rate, and physiological changes (e.g why early lethal if it was, which was mentioned in the discussion). The work also lacks novel mechanistic insights into how GNAQ cause vascular malformations and solely selected ERK1/2 for study. Therefore, the study was considered as mouse model with phenotypic verifications. R: We thank the Reviewer for this feedback. 1-We have extensively revised our manuscript and now include data on the EC-specific recombination in pups and adult mice (see Suppl. Fig. S2 and S8). 2-We provide brightfield and immunohistochemistry/immunofluorescence images for both subcutaneous and intestinal muscularis tissue for all of the relevant experiments (see Figs.1,2,7). 3-In this substantially revised manuscript we have characterized vascular morphogenesis defects (Figs.1-2, Suppl. Videos 1 and 2), increased EC proliferation (Fig.2, Suppl. Video 2), vascular permeability (Fig.3, Suppl. Video 3), coagulopathy (Fig.4, Suppl. Video 4), transcriptomic analysis of the GNAQ-mutant EC (Fig.5 Suppl. Fig.S14) and MAPK signaling in EC (Fig.5,6). Furthermore, we have extended our characterization to include Kaplan-Meier survival curves, and weight curves (see Figs.1,2,7 and Suppl. Figs.S4, S6, S18). 4-We have now included transcriptomic analysis of the GNAQ-mutant EC and showed that the MAPK signaling pathway is one of the top differentially regulated KEGG pathway (Fig.5). The transcriptomic analysis also highlighted upregulation of genes implicated in angiogenesis, inflammation and complement and coagulation cascades. Up to date, the mechanisms by which Gαq regulates aberrant vascular development and remodeling remained unknown, this manuscript provides a murine model for studying (EC-specific) GNAQ-related vascular tumors and related severe complications including hyperpermeability and severe coagulopathy such as KMP, these will aid in development of targeted therapies, which are highly needed to treat young patients and currently lacking.
The phenotype characterization: it was limited to a very short time frame (  Section imaging of retinas and hyaloid would have better visualize the tufts. Vessel diameters: were they artery, vein or microvessels? Were microphage number differed in KO groups vs WT? R: We have tried sectioning the retinas as suggested, however it is difficult to find the exact location of the tufts and we believe the flat mount is the best approach to visualize them. To investigate the tuft location (artery versus vein-capillary), we have used the retina as model in which arteries and veins can be readily distinguished. Analysis of mutant iEC-GNAQ Q209L mice at P8 showed that vascular tufts were exclusively localized in retinal veins and capillaries, while absent in the arteries (see new Suppl. Fig.S5). We did not analyze macrophage number as we feel it is outside the scope of this study. Hyaloid data were removed based on concerns raised by Reviewer 1 that the phenotype could reflect lack of hyaloid vessel regression rather than increased vascularity. Some tissues were examined (from Fig.1-5). However, the study was simply vascular densities. No functional studies were performed. R: In this revised manuscript, we included new data on functional studies to evaluate proliferation (Fig.2H-J, Suppl.  Fig.S9, Suppl. Video 2), vascular permeability (Fig.3, Suppl. Video 3), coagulopathy (Fig.4, Suppl. Figs.S10-12, Suppl.  Video 4). Furthermore, all these functional parameters are now evaluated in mice treated with Trametinib (Fig.7) Fig.6 had human speciesmen were strength.
R: Thank you for the positive comment! Fig.7 therapeutics with MEK inhibitor was nice. It would be more informative to dissect why ERK1 and if GNAQ inhibitors had better efficacy in the mouse models.
R: Thank you for the positive comment. In this revised manuscript we include transcriptomic and western blot data supporting the importance of the MAPK pathway in GNAQ mutant EC and we included analysis of the transcriptional changes driven by Trametinib. We additionally included data on a therapeutic study with the use of Trametinib on established lesions, and report extended mutant mouse survival compared to vehicle treated animals (Fig. 7N).

Reviewer #3 (Remarks to the Author):
In this manuscript, Schrenk et al. generated the first murine model for GNAQ-related vascular anomalies, exhibiting endothelial cell (EC)-specific expression of the constitutively active mutant GNAQ pQ.209L of the Gαq protein. Gαq gain-of-function mutation results in widespread abnormal vascular morphogenesis and potential thrombocytopenia and severe coagulopathy. Such vascular morphogenesis defects are consistent with findings in patients with Gαq gain-of-function mutations. The authors characterized vascular abnormalities in the brain, retina, hyaloid, subcutaneous muscle and intestinal muscle in GNAQQ209 mice at different time points of development, and confirmed MAPK/ERK activation and proliferation markers in patient-derived tissue. Finally, the authors performed proof-of-concept experiments, demonstrating that MEK inhibition with Trametinib rescued vascular and coagulopathy phenotypes.
Since mechanisms by which Gαq regulates aberrant vascular development and remodeling remain unknown, this paper provides a murine model for studying (EC-specific) GNAQ-related vascular anomalies that will aid in development of targeted therapies, which are currently lacking. The authors provide extensive evidence of aberrant vascular morphologies that recapitulate what are seen in patients. The manuscript is easy to follow, and figures containing schematics of mouse breeding and experimental timeline are helpful. However, there are important limitations in this manuscript that limit enthusiasm that are listed below as well as more minor concerns. R: As suggested, we have extended our analysis of the mutant vasculature functionality. In this revised manuscript we have characterized 1-vascular morphogenesis defects ( Fig.1-2, Suppl. Video 1), 2-proliferation (Fig.2, Suppl. Videos 2), 3-increased permeability (Fig.3, Suppl. Video 3), 4-coagulopathy (Fig.4, Suppl. Video 4) and 5transcriptomic analysis of the GNAQ-mutant EC and EC pro-inflammatory phenotype (Fig.5). Furthermore, we have extended our characterization to include Kaplan-Meier survival curves (see Fig.1,2,7 and Suppl. Figs.S4, S6, S18).
2. There are very important controls not done in this manuscript that are needed to complete the claim that an ECspecific overexpression of GαqQ209 is responsible for the observed vascular effects. These include showing that there was no leakage of expression until tamoxifen was given. The controls in Figs 2-8 were done in mice that were littermates but did not have the GαqQ209 expression vector. It should have been done in mice with the GαqQ209 construct but without tamoxifen. Were the vessels normal until that was done? Also were the non-ECs normal and the effect was targeted to the endothelium? Finally, what was the relative level of GαqQ209 pre and post tamoxifen versus native Gαq? Was the effect seen just because there is a massive increase in Gαq in the ECs and even normal Gαq would have resulted in the same outcome.
R: We thank the reviewer for these comments and insights. 1. As noted in response to R1 and R2, we now included no-tamoxifen mutant mice controls for most of the experiments and highlighted the specific genotype of mice used for every graph. In all of our experiments, in both pups and young adults no-tamoxifen mutant mice had normal vascular phenotype, comparable to tamoxifen treated controls or WT mice. Additionally, we have included comprehensive analysis in pups and young adult animals to test the efficiency and specificity of the Cdh5-iCreER driver with and without tamoxifen, by crossing GNAQ Q209L mice with the Rosa26 tdTomato lineage reporter. There was only minimal recombination in Chd5-iCreERT2; tdTomato mice that did not receive tamoxifen (1.8±088% in pups; 2.7±2.50% and 2.20±1.60% in adult mice subcutaneous and intestinal muscularis tissues; see Suppl. Figs.S2 and S8). In addition, we did not detect tdTomato expression in non-EC. Notamoxifen mutant mice are alive at 13 months, and do not have overt phenotype or illness. 2. This is an important point. To assess that the phenotype is driven by the mutant GNAQ Q209L expression and not just GNAQ WT overexpression we have performed transcriptomic analysis of EC overexpressing GNAQ-Q209L and GNAQ-WT. The principal component analysis in Fig.5A shows that EC GNAQ-WT + Dox clusters very closely to GNAQ-WT-no-Dox, while there was a clear separation from the mutant EC GNAQ-Q209L + Dox. Furthermore, we also show in Fig. 5F that increased p-ERK is exquisitely upregulated in a Doxycycline-dependent manner in EC expressing GNAQ-Q209L, while no upregulation was noted in EC overexpressing GNAQ-WT.
3. There were two different EC-specific promoters studied. The rationale for that and why some studies were done mixing the two or with only one is not explained other than one statement that the results were not different, yet we don't know efficiency of the two in driving human GαqQ209 expression in different tissues.
R: Initial experiments in pups were performed with the Pdgfb-iCreER T2 , however due to the COVID pandemic we had to cut down the number of mouse lines and switched to the Cdh5-iCreER T2 . We have revised the manuscript and now included in the main text only data obtained with the Cdh5-iCreER T2 . In Suppl. Fig. S7 we show that vascular density, area and vessel diameter in the subcutaneous and intestinal muscularis tissues are comparable in Pdgfb-iCreER T2 and Cdh5-iCreER T2 -driven GNAQ mutant mice.
MINOR CONCERNS 1. The authors should discuss Huang et al. ATVB 2022. (PMID: 34670408) on EC biology and its support of the studies in this manuscript. R: We now include this important reference.